A bioreactor can be defined as a vessel in which a biological conversion is effected. This definition can apply to any conversion involving enzymes, micro-organisms, or animal or plant cells. However, we will use the word bioreactor to refer to vessels (usually made of glass, metal or a polymer) in which organisms are cultivated in a controlled manner. Modern cell cultivation is typically accomplished using a bioreactor or a fermentor vessel. Despite the fact that a bioreactor and a fermentor are essentially similar in design and general function, the dichotomy in nomenclature is sometimes used to distinguish between animal and plant cell culture. Herein we will use the term bioreactor in an inclusive, generic sense as including both aerobic and anerobic cultivation of both microbial, animal and plant cells, and thus encompassing a fermentor.
The goal of an effective bioreactor is to control, contain and positively influence a desired biological reaction. One desired biological reaction considered here is the growth of microorganisms. The most popular method for accomplishing this is a batch cultivation system. See, for example, James Lee, Biochemical Engineering, Washington State University, e-book, 2002. For simplicity and clarity we will detail a batch biological process here, but it is to be understood that the port design of the present invention is applicable to any bioreactor process (e.g.: perfusion or other continuous process) and also to a chemical process where monitoring of the reaction vessel is important.
In a batch biological process the microorganisms are inoculated into a culture medium and the growth cycle commences. This growth cycle consists of:    1. Lag phase: A period of time during which the cells have not yet commenced significant growth.    2. Accelerated growth phase: The period during which the number of cells increases and the cell division rate reaches a maximum.    3. Exponential growth phase: The period during which the number of cells increases exponentially as the cells divide. The growth rate is increasing during this phase, but the division rate is constant and at its maximum.    4. Decelerated growth phase: After the growth rate reaches a maximum it is followed by a deceleration in both the growth rate and division rate.    5. Stationary phase: The cell population reaches a maximum value and does not increase further.    6. Death phase: After the nutrients available for the cells are depleted and/or the bioreactor environment becomes too hostile, cells will start to die and the number of viable cells will decrease.
Generally, it is desirable to minimize the lag phase, ensure adequate nutrient concentration (e.g.: glucose) in the growth phases, and to delay the onset of the cell death phase as long as possible. In order to accomplish this optimization of the biological reaction, i.e., specifically in order to maximize the yield of product from a bioreactor, two factors must be considered. The first is providing suitable reactor design parameters for the particular biological, chemical, and physical aspects of the system. The second area of major importance in bioreactor design is monitoring the conditions inside the reactor so as to control the parameters relevant to micro-organism growth. These parameters can include, but are not necessarily limited to:    1. Temperature    2. pH    3. Dissolved gasses (e.g.: O2, CO2,)    4. Nutrients (e.g.: sugars such as glucose, proteins, amino acids, fats)    5. Inorganic Salts    6. Water and foam levels in the bioreactor    7. Product formation (e.g., cholesterol, vitamins)    8. By-product removal (e.g.: lactic acid, NH3)    9. End products: e.g., enzymes and proteins    10. Cell density (concentration)    11. Cell viability    12. Conductivity and osmolality
It should be noted that many of these parameters are critical irrespective of which method of cultivation (i.e., batch, continuous, or perfusion) is used, and that some parameters are more important than others depending on the type of microbe or cell being grown. The process can, for example, be a batch process in a stirred tank, a perfusion process or can utilize an airlift bioreactor etc. However, knowledge of the parameters that relate to cell growth, cell respiration, cell death, and yield maximization, are critical to understanding and optimizing any growth process.
Due to the requirements for handling hydrostatic pressure and the pressure associated with cleaning/sterilization procedures, bioreactors have heretofore generally been constructed of stainless steel such as 316L, sometimes including glass components. The initial cost of a 316L stainless steel bioreactor and the concomitant plumbing is substantial. The cost to run the impeller, the aerator, and to cool/heat the bioreactor is also sizable. Finally, the costs of cleaning and sterilizing such a reactor after use, and disposing of the waste water from the cleaning process are non-negligible. Given the aforementioned costs associated with running a conventional steel bioreactor, many organizations have begun using disposable bioreactors. These single use bioreactors are generally constructed using films which have been proven to be biocompatible and animal derived component free. Often these films must comply with standards set by both the U.S. Food and Drug Administration (FDA) and the United States Pharmacopeia (USP). The layer in contact with the media is often polyethylene or ethylene-vinyl acetate (EVA), and the outer (reinforcing) layer is often nylon, though many different combinations of materials and construction techniques have been used or, at least, proposed. Variations in the materials utilized are frequently determined by the precise mixing implementation, and/or on whether or not the bag needs to be gas permeable. Often these disposable bag reactors include gas inlet and outlet ports, inlet and outlet filters, a pressure control valve, and a port to fill the bag with aqueous media.
Given the many variations in disposable bioreactor design, there are also many variations in the methods for accomplishing mixing and aeration. One popular design for bag style bioreactors uses a platform that implements a rocking motion to the bag in order to mix and aerate the bag contents (see e.g., Published U.S. Pat. No. 6,190,913). Other disposable bioreactor designs look more like conventional stainless steel and glass reactors, and use impellers that essentially mimic the mixing methods used in conventional steel and glass bioreactors (see for example U.S. Patent Application 2005/0239199A1). Many claims are made on both sides of this issue regarding the efficacy of the various mixing methods. The rocking motion allegedly is gentler than the use of impellers and therefore leads to less damage to mammalian cells but apparently can stress the seams of the bag and may not always provide sufficiently effective mixing and/or oxygenation. The impeller based designs are generally more effective agitators, but can sometimes damage cells, and also require more infra-structure.
Irrespective of the method used for mixing in disposable bioreactors, it is clear that suitable methods for determining the efficacy of the bioreactor process and reliable methods for obtaining timely information on the status of the process are lacking. Only recently have many of the leading disposable bioreactor manufacturers attempted to marry sensor technology with their bag. The general ambiguity regarding the efficacy of the mixing and oxygenation in disposable bioreactor bags highlights the fundamental need to be able to monitor critical process parameters. Current practice with standard glass and steel bioreactors is to introduce the applicable probes through a port in the reactor wall or head plate. The use of such a port allows the calibration and testing of the probes before placing the probes into the bioreactor. However, with disposable bioreactors it is difficult to measure many of the aforementioned critical parameters using most of the currently available probes due to the dielectric nature of disposable bioreactor bags. Specifically, most existing electrochemical probes used to measure reaction parameters such as dissolved oxygen, pH, and dissolved CO2 require the probe to be both shielded and grounded. Achieving this is often complicated by the lack of a metal or at least a conductive housing for a disposable (and hence dielectric) bag bioreactor. Additionally, many of the traditional probes are too large or otherwise not functionally useable in disposable bag bioreactors.
One method of avoiding many of the issues inherent with traditional electrochemically based sensors in polymeric bioreactor bags is through the use of different transduction methodologies. For example, probes for measuring dissolved oxygen, pH, and dissolved CO2 utilizing optical based sensing technologies have recently become available. Different methods have been reported for integrating the optical sensors in a disposable bioreactor bag. One possible method is to seal a patch having a probe mounted on it directly into the bag (See Published U.S. Patent Application: 2005/0272146). Shown in FIG. 1 is a disposable bag as described in the above-cited patent application, where 206 is the location for the probes which are to be attached to the inside of the bag. The patch needs to be assembled into the bag using bio-compatible materials which can endure gamma radiation or alternative methods of sterilization. Though not entirely clear from the Figures of the above-cited patent application, it is possible to mount “dots” containing a dye sensitive to a target analyte (e.g., pH, or dissolved O2 or CO2,) on the inside surface of a patch of bio-compatible plastic and use an RF or thermal source to fuse this patch into the bag wall. The dots on this patch can contain various fluorescent materials for use in sensing pH, dissolved O2, and dissolved CO2. These dots generally utilize a “dye”e.g., a transition metal complex whose fluorescence lifetime is quenched by the presence of the target analyte to be measured. The material can be printed onto the patch or attached using a decal. The only requirements are that sufficient light to excite the dye present on the inside surface of the patch is transmitted through the patch window, and that sufficient fluorescence signal is transmitted back out through the window such that the decay in the signal strength can be monitored from outside the bag. The monitoring or interpretation of the signal is typically done at the transmitter which houses the user interface, the signal processing, and in some instances the opto-electronic components of the analyte monitoring system.
In the case of disposable bag reactors utilizing a patch of material fused into the lining, or simply using the disposable reactor lining itself to mount optically based probes significant limitations arise in that it is difficult to bring high fidelity optical probes or intrusive optical connections into and out of the bag. Using a simple dot affixed to the inside of the disposable bioreactor wall, only optical signals of limited fidelity can be transmitted back and forth. This puts greater requirements and restrictions on the optics and electronics in the transmitter. Furthermore, the materials used to make the bags are typically chosen for bio-compatibility, gas permeability, and strength, rather than for optical transparency. This makes bringing in high fidelity optical signals problematic as the light may be attenuated and/or randomly scattered as it passes through the wall of the bag. It also makes it difficult or even impossible to optimize the efficiency of the optical delivery and collection system.
Other issues which arise from relying on the relatively simple technique of affixing a sensor material to the inside of a disposable bioreactor bag wall are that it can preclude the installation of more advanced sensors. Specifically, as sensors continue to advance it is clear that signals in addition to an optical signal will need to be passed into and out of the bioreactor. It is likely that when sensors utilize optical, electrical, chemical, acoustic, magnetic, and micro-fluidic technologies or a subset of these technologies, more communication with outside instrumentation will be necessary than is possible using currently available techniques.
Through the use of a more sophisticated interface or window into the bioreactor, multiple measurements can be simultaneously accomplished. These measurements can include any of the parameters discussed above. The measurement methods include, but are not limited to those that are based on optical, electrical, chemical, bio-chemical, acoustic, magnetic, and micro-fluidic techniques, or any combination thereof. These techniques can either measure the headspace gas or liquid medium inside the disposable bioreactor. Additionally, these measurements can utilize any part of the electromagnetic spectrum including ultra-violet, visible, near infrared, and mid infrared to far infrared radiation or RF and DC electrical fields to probe the biochemical or chemical system inside the reactor bag. Optically, the measurements can also be done using Raman Stokes or anti-Stokes radiation, using FTIR methods, auto-fluorescence, photo-acoustic or near field optical systems to obtain the data of interest. Other measurements can involve miniaturized or on-chip: mass spectrometry, liquid chromatography, flow cytometry, or nuclear magnetic resonance (NMR). This data can be directly or indirectly indicative of the state of the bioreactor medium. Virtually any transduction method that can be used with the port of the present invention, and can be directly correlated to an analyte of interest allows for a useful sensor.
In addition to more traditional disposable bag bioreactors having rockers (Wave Biotech) or mixers (Hyclone, XCellerex) or spargers (e.g., air-lift) for agitation, other types of bioreactors including those disposable bioreactors that utilize hollow fibers, or parallel plates for growth (e.g.: Corning Cell Cube®) can benefit from the use of the port and disposable probe technology of the present invention. Finally, given that our technology is conceptually also compatible with standard ports on glass and metal bioreactors, the present invention can also be utilized with non-disposable bioreactors.